1. Field of the Invention
The present invention relates generally to the fields of protein chemistry and biochemical assays and reagents. More specifically, the present invention relates to modified fluorescent proteins and to methods for their use.
2. Description of the Related Art
The Green Fluorescent Protein (GFP) from the jellyfish Aequorea Victoria has been widely used as a reporter in the determination of gene expression and protein localization (1-4). GFP cDNA can be expressed in various cells or organisms with an easily detected fluorescence in the absence of any substrate or cofactor (5).
GFP is a 27-kDa, single-chain polypeptide of 238 amino acids (6). A key sequence of Ser-Tyr-Gly at amino acids 65 to 67 near the N terminus functions as the GFP fluorophore (7). These three amino acids undergo spontaneous oxidation to form a cyclized chromophore responsible for the fluorescence of GFP (8). Enhanced GFP (EGFP) is a mutant of GFP with a 35-fold increase in fluorescence (9-11). This variant has mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64, and is encoded by a gene with optimized human codons (10).
Because of its easily detectable green fluorescence, green fluorescent protein from the jellyfish Aequorea Victoria has been used widely to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in numerous species and in a wide variety of cells. GFP is a very stable protein, and can accumulate; thus, GFP is often toxic to mammalian cells.
Recently, crystallographic structures of wild-type GFP and the mutant GFP S65T reveal that the GFP tertiary structure resembles a barrel (Ormo et al. (1996) Science 273: 1392-1395; Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nature Biotech 14: 1246-1251). The barrel consists of beta sheets in a compact antiparallel structure. In the center of the barrel, an alpha helix containing the chromophore is shielded by the barrel. The compact structure makes GFP very stable under diverse and/or harsh conditions, such as protease treatment.
A great deal of research is being performed currently to improve the properties of GFP and to produce GFP reagents useful for a variety of research purposes. New versions of GFP have been developed via mutation, including a "humanized" GFP DNA, the protein product of which enjoys increased synthesis and improved folding in mammalian cells (see Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) Gene 173, 33-38; Haas, J., Park., E. C., and Seed, B. (1996) Current Biology 6, 315-324; and Yang, T. T., Cheng, L., Kain, S. R. (1996) Nucleic Acids Research 24, 4592-4593). One such humanized protein is "enhanced green fluorescent protein" (EGFP). Other mutations to GFP have resulted in blue- and red-fluorescent light emitting versions.
Cells are capable of responding to various stimuli by activating or repressing the expression of particular genes whose products exert a wide range of effects on biological processes. Such stimuli include heat shock, steroid hormone, growth factors, cytokines, etc. The process of gene transcription itself is the major point for regulation, although gene expression can be regulated at the post-transcriptional level. The external signals or stimuli may affect gene expression via regulation of transcriptional factors in a process called signal transduction. External signals exert influence by, for example, inducing conformational changes in these factors, modifying them chemically, or directing formation of ligand/protein complexes. The dissection of signal transduction pathways provide important information for the drug discovery and design.
A number of important signal transduction pathways require the activation of transcription factor NF-kB. Compounds affecting NF-kB activation includetumor necrosis factor .alpha. (TNF.alpha.), interleukin 1.beta. (IL-1), liposaccharide (LPS), and phorbel ester (PMA). The activation of NF-kB leads to induction of a large array of genes whose products contribute diverse important biological processes, such as cell growth, apoptosis, inflammation, and immune responses. NFkB is the prototype of a family of dimeric transcription factors. It consists of p50(NFkB1), p52(NFkB2), RelA(p65), RelB, c-Rel. The Rel/NFkB family of proteins share an approxomately 300-amino acid Rel region that binds to DNA, interacts with each other, and binds its inhibitor, IkB. There are at least six species IkB proteins that are identified to associate with NFkB/Rel proteins. All IkB proteins have 5-7 ankyrin repeat domains, each with a length of about 30 amino acids, that interact with the NFkB Rel region.
The mechanism of NFkB activation has been well documented. NFkB is present in an inactive form in the cytoplasm where it is bound to IKB. Cellular activation in response to a variety of inducers leads to the rapid release of NFkB from IkB. This activation is independent on protein synthesis. In response to diverse stimuli, an intracellular cascade of protein kinase activity is triggered, and IkB is phosphorylated at two serines (Ser 32 and Ser 36) specifically and degraded rapidly by 26S proteasome. Uncomplexed NFkB rapidly translocates to the nucleus where transcriptional activation of gene expression occurs within minutes.
The search for agonists or antagonists that can activate or inhibit NFkB are major drug targets for pharmaceutical companies. Two very common methods used for studying the activation of gene expression are the in vitro gel shift assay or assays involving in vivo transcriptional induction of a reporter. Because the mechanism of NFicB activation is well defined, a number of assays based on the activation pathway have been designed and used to monitor NFkB-induced gene expression. The assays include measuring phosphorylation of IkB, degradation of IkB, and ubiquitination of IkB. These assays are often used in the research labs; however, for large scale drug screening, the assays are either time consuming or tedious.
The prior art is deficient in an methods and research tools for easy, high throughput assays for measuring degradation of IkB. The present invention satisfies this long-standing need in the art.